The current phase I R21 study has accomplished three Specific Aims. The feasibility of retrieval/extraction of protein, RNA, and DNA from archival formalin-paraffin tissues by modified Antigen Retrieval (AR) methods was demonstrated. PCR based methods were adapted and applied successfully for the amplification and quantitation of RNA/DNA extracted by modified AR from formalin-paraffin tissues. Three model systems were developed to monitor the efficacy of the modified AR process, including novel simulated or 'faux tissues' and purified 'protein matrix pellets'. Continuation of these studies in this R33 proposal focuses upon the incorporation of these advances into an 'integrated systems approach' to the molecular analysis of cancer tissues that have been preserved as formalin-paraffin blocks. Currently, immunohistochemical (IHC), in situ hybridization (ISH) and PCR based assays are widely applied to formalin-paraffin cancer tissues in 'routine diagnosis' and in basic cancer research, but the reproducibility and the validity of the findings are open to serious question due to the lack of uniform methods, in particular the total lack of 'standard reference materials', which are considered essential to the use of analogous methods in the diagnostic clinical laboratory. This proposal addresses pre-analytical, analytical, and post-analytical aspects of the use of IHC, ISH and PCR based assays as applied to formalin-paraffin cancer tissues. Integral to this approach is the development of reference standards for key analytes that are, or may be, employed as cancer or prognostic markers, against which results obtained by the analysis of formalin-paraffin cancer tissues will be calibrated to yield strict quantitative findings for diagnostic and research purposes. To meet the goal of objective quantitation in tissue sections, spectral image analysis will be performed at the USC Image Core, for which we have shown the feasibility of quantitative analysis of up to four different analytes simultaneously in the same formalin-paraffin section. This proposal is multi-disciplinary, including collaborations with the USC Cellular Imaging Core at Childrens Hospital of Los Angeles, the Huntington Medical Research Institute in Pasadena, Roche Molecular Systems Inc., the National Institute of Standards and Technology (NIST), and the University of California, San Francisco.